We investigated whether knockdown of Snail expression with siRNA would prevent alcohol-stimulated EMT (vimentin expression). Caco-2 cells were treated with alcohol (0.2%, 43 mM) for 4 days. Some cells were pretreated (48h) with an irrelevant siRNA control or siRNA (100nM final) specific for Snail as described (Forsyth et al., 2007). Western blots were used to assess Snail and vimentin protein expression. As seen in Figure 5, treatment with alcohol caused an increase in vimentin expression in media treated as well as control siRNA treated cells (lanes 2&3). However, Snail knockdown (48% mean knockdown) with specific siRNA prevented alcohol-induced vimentin expression (lane 4; p≤.05 vs. siRNA control, t-test). Note that these data are for total Snail only to assess knockdown. Our previous data (Figure 3A–D) show alcohol stimulates nuclear localization of Ser246-phosphorylated Snail.
